The expression of the key virulence factors of Bacillus anthracis are tightly regulated, responding to the presence of carbon dioxide, which is a signal that the bacteria are in a mammalian host. A key regulator of the response to carbon dioxide is the AtxA protein. We have previously characterized the function of atxA using Next Generation sequencing. In work done during 2016, we have extended study of AtxA by constructing plasmids having different sets of nucleotides from the original pagA promoter. Using qRT-PCR approach for pagA transcription under control of these sequences we hope to identify parts of pagA promoter responsible for AtxA binding. In addition to providing information about the normal role of AtxA, this work may lead to host strains and plasmids that support higher levels of protein expression of heterologous proteins in multi-protease deficient B. anthracis strains. In a second project executed in 2016, genes and sequences needed for maintenance of the key virulence plasmid pXO1 were further characterized. Two genetic tools developed for DNA manipulation in B. anthracis (Cre/loxP and Flp/FRT systems) were used to identify pXO1 regions required for plasmid stability. Analysis identified three genes that are necessary for pXO1 maintenance: tubZ (GBAA_RS28715), tubR (GBAA_RS28720), and intXO (GBAA_RS29165). Continuing analysis of the proteins encoded by these genes may allow development of anti-infective agents - those that do not directly kill the pathogen but instead render it less virulent, thereby allowing host immune responses to effectively combat it. The intXO gene encodes a tyrosine recombinase, along with its adjacent 37-bp perfect stem-loop (PSL) target. We showed that the newly discovered site-specific IntXO-PSL recombinase system that contributes to the maintenance of the B. anthracis plasmid pXO1 can be used for genome engineering (editing) in a manner similar to that of the Cre-loxP and Flp-FRT systems. In a collaborative study published in 2016, evidence was obtained that B. anthracis immune inhibitor A (InhA)-type metallopeptidases are potential virulence factors secreted by members of the Bacillus cereus group. Two paralogs from anthrax-causing B. anthracis (InhA1 and InhA2) were shown to degrade host tissue proteins with broad substrate specificity. Analysis of their activation mechanism and the crystal structure of a zymogenic InhA2 variant revealed a 750-residue four-domain structure featuring a pro-peptide, a catalytic domain, a domain reminiscent of viral envelope glycoproteins, and a MAM domain grafted into the latter. This domain, previously found only in eukaryotes, is required for proper protein expression in B. anthracis and possesses considerable flexibility. Latency is uniquely modulated by the N-terminal segment of the pro-peptide, which binds the catalytic zinc through its -amino group and occupies the primed side of the active-site cleft. The present results further our understanding of the modus operandi of a B. anthracis secretome regulator.